The brain detects alterations in diet and energy balance, and through various neural circuits, regulates energy intake and energy expenditure. Dysfunction of these homeostatic mechanisms results in obesity, an epidemic problem in affluent societies. Identification of the neural circuits, as well as the molecular effecters operating within these circuits, is a major focus of obesity research. The last decade has seen the discovery of a number of proteins that are critical components of this system. 1 such protein is the meIanocortin-4-receptor (MC4R), a G-protein-coupled receptor expressed by a number of neurons in the brain. The essential role of MC4Rs is evident from the presence of severe obesity in both gene knockout mice and in humans with naturally occurring mutations. In the case of Mc4r null mice, in which energy balance has been studied in great detail, obesity has been found to be caused by the combined effects of increased food intake (hyperphagia) and decreased energy expenditure. While the importance of MC4R signaling is clear, the neural substrate responsible for MC4R-mediated regulation of food intake and energy expenditure is presently unknown. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB-Mc4r) that can be re-activated by Cre-recombinase (Balthasar et al., Cell, IN PRESS). Mice homozygous for the loxTB-Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that there is disassociation in pathways controlling energy balance and that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. In work proposed in the present application, we will use loxTB-Mc4r mice, as well as Iox-Mc4r mice, in combination with various transgenic mice expressing Cre-recombinase in different, potentially important groups of neurons, to identify the specific neurons that mediate MC4R's effects on food intake and energy expenditure. This information will facilitate the construction of a wiring diagram of energy homeostasis.